Recombinant Production of an Inulinase in a Saccharomyces cerevisiae gal80 Strain

Abstract

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 was overexpressed by using the GAL10 promotor in a ∆gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to a mating factor α signal sequence for secretory expression. Use of the ∆gal80 strain allowed for the galactose-free induction of inulinase expression using a glucose-only medium. Shake-flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the ∆gal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both aerobic and anaerobic conditions by about 2.9-and 1.7fold, respectively. A 5-l fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at the OD 600 of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5-l scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and with the pH controlled at 5.0. The temperature was maintained at 30 o C and 37 o C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/l, respectively.

Enhanced expression of the codon-optimized exo-inulinase gene from the yeast Meyerozyma guilliermondii in Saccharomyces sp. W0 and bioethanol production from inulin

“…It has been found that the engineered strains of S. cerevisiae carrying the exo-inulinase gene show a higher inulinase activity than those carrying the endo-inulinase gene. For example, the exo-inulinase gene from K. marxianus NCYC2887 was overexpressed by using the GAL10 promoter in a S. cerevisiae △gal80 strain, and the recombinant inulinase activity was 34.6 U/mL (Lim et al 2010). Moreover, our recent results have indicated that after INU1 from M. guilliermondii strain 1 was actively expressed in Saccharomyces sp.…”

Engineering an industrial Saccharomyces cerevisiae strain with the inulinase gene for more efficient ethanol production from Jerusalem artichoke tubers

“…Engineering S. cerevisiae strains with heterologous inulinase genes to enhance inulinase production has been explored by plasmid transformation . However, selective pressure is required for recombinants generated by this strategy, which is not practical for industrial application.…”

High-level secretory expression and characterization of the recombinant Kluyveromyces marxianus inulinase

“…KmINU production using K. marxianus ATCC 16045 under various culture conditions has been tested, and a maximal yield of 208 U/ml has been achieved [24]. To improve inulinase production, heterologous expression systems have been tested [10][11][12][25][26][27][28]. However, the specific activities and production titres of those products remained low (Table 2), which could be a major obstacle for the large-scale production and use of inulinase.…”